Polyepitopic proteinic fragments of the E6 and E7 HPV proteins, production and use thereof in vaccines

ABSTRACT

Polyepitopic fragments of the E6 or E7 protein of HPV comprise a peptide sequence of about 15 to 30 amino acids. This peptide sequence contains amino acid sequences of at least 3 different epitopes binding stably to HLA molecules of identical or different type, when these epitopes are obtained by enzymatic degradation of the peptide sequence, particularly in the proteasome, such that at least 4 HLA molecules of different types bind to these epitopes. These 4 HLA molecules are selected from among those of types A1, A2, A3, A11, A24, A29, B7, B8, B18, B27, B35, B44, B51 and B62.

BACKGROUND OF THE INVENTION

The present invention has for its object polyepitopic protein fragments,such as those of the E6 and E7 proteins of human papillomavirus, or ofthe human p53 protein, their process of production, and their uses,particularly in the field of therapeutic or preventive vaccination.

SUMMARY OF THE INVENTION

The invention more particularly has for its object the use ofpolyepitopic fragments of a predetermined protein for the preparation ofmedications adapted for the prevention or treatment of pathologies inwhich said protein is recognized by the cellular immune system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an E6 protein of HPV 16;

FIG. 2 shows an E7 protein of HPV 16.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, said polyepitopic fragments are such that their N-terminalamino acid corresponds to the N-terminal amino acid of the epitopelocated upstream of one or several other epitopes of a polyepitopicregion of said protein, and their C-terminal amino acid corresponds tothe C-terminal amino acid of the epitope located downstream of theabove-mentioned epitope or epitopes of said polyepitopic region.

Thus, the above-mentioned polyepitopic protein fragments of the presentinvention correspond preferably to the polyepitopic regions of apredetermined protein, namely to the regions containing several epitopesrecognized by the T cells in association with the different molecules ofthe major complex of histocompatibility (MCH), said regions beingselected from those having the characteristic of being degraded in vitroin shorter peptides by proteasomes, such as the 20S proteasome, when theprotein fragment tested is placed in the presence of said proteasome,particularly according to the following detailed method. The proteinfragment (about 75 μg when it is a polypeptide of about 30 amino acids)is incubated at 37° C. with about 15 μg of 20S proteasome (CalbiochemRef 539150, La Jolla, Calif., USA) in 500 μl of the following buffer: 20mM Tris-HCl pH8, 0.5 mM EDTA. Aliquots of 50 μl are removed afterincubation times of 24 and 48 hours, and are analyzed by high pressureliquid chromatography (HPLC). The digestion products of the proteasomesare separated by RP-HPLC (Perlin Elmer) by using a C18 column and anacetonitrile gradient (from 0 to 100% containing 0.1% trifluoroaceticacid, for 90 minutes, elution rate 0.8 mL/min). The cleavage productsare detected at 214 nm by an absorption detector (759A, AppliedBiosystems).

Preferably, the polyepitopic regions defined above have thecharacteristic of containing hydrophobic amino acids.

The different epitopes of the polyepitopic region of the predeterminedprotein, and delimiting the epitopic protein fragments, are preferablyselected from the peptides;

binding to a predetermined molecule of MCH, particularly to a moleculeof the predetermined HLA type, and this from concentrations of about10^(−≢)M to about 10⁻¹⁰ M in peptide for concentrations of about 10⁻⁷ Mof HLA molecules, particularly under the conditions described hereafter,

and forming a stable complex with said MCH molecule, namely particularlya complex in which said peptide remains bound to said molecule for atleast about 3 hours at 37° C.

By way of illustration, the above-mentioned epitopes of the inventionare selected from among the peptides adapted:

on the one hand of associating with the molecules of MCH, particularlyby using the following method:

incubation (particularly for about 2 hours at 25° C., then about 15hours at 4° C.) of the peptide in the presence of MCH molecules, fromthe lysis of human or animal cells, or purified particularly by affinitychromatography from human or animal cell lines,

trapping complexes formed during the preceding step on a solid supportcovered with a first antibody, particularly monoclonal, recognizingspecifically the CMH molecules in their configuration dependent on theirconnection to said peptide,

addition to the preceding solid support of a second marked antibody,particularly by coupling to a radioactive, enzymatic or fluorescentmarker, said marked antibody recognizing specifically either the longchains of MCH in their configuration dependent on their connection tothe peptide, or the short chain of MCH or the β2-microglobulin bindingspecifically to the different long chains of the MCH in theirabove-mentioned configuration,

detection, after rinsing of the solid support, of the possible presenceof the second marked antibody remaining fixed on the solid support,signifying an association effect between the molecules of MCH and thepeptide studied,

and, on the other hand, forming a complex with said MCH molecules, whosestability can be evaluated by the use of a method of following accordingto time the connection established between the peptide and the MCHmolecules, this method being preferably carried out according to aprotocol identical to the preceding method, but in which the incubationstep of the peptide in the presence of MCH molecules on the solidsupport covered with said first antibody, is preceded by a preliminarystep of eliminating the free peptide adapted that may be present in thereaction medium, particularly by washing the solid support, saidincubation step being carried out (preferably at a temperature of 37°C.) for variable times of 1 hours, 3 hours, 5 hours, 24 hours and 48hours.

As mentioned above, the epitopes of the invention should be recognizedby the T cells in association with the MCH molecules and associate withthese latter, particularly in the framework of the practice of therecognition test described above. This association can be weak(detectable at concentrations of peptide analogs of the order of 10⁻⁴ to10⁻⁵ M), intermediate (detectable at concentrations of peptide analogsof the order of 10⁻⁶ to 10⁻⁷ M), or strong (detectable at concentrationsof peptide analogs of the order of 10⁻⁸ to 10⁻⁹ M). The peptidesassociated with the MCH molecules in the scope of the present inventionare preferably adapted to bond during at least about 3 hours, to saidMCH molecules.

The invention more particularly has for its object the epitopes (alsodesignated peptides above and hereafter) as described above andcharacterized in that they are selected from among those adapted:

to induce in vitro cytolysis by cytotoxic T lymphocytes, of target cellshaving at their surface the above-mentioned peptide associated with theMCH molecules, said cytotoxic T lymphocytes being preferably removedfrom a patient having a pathology in which the peptide studied isimplied,

and inducing in vitro the secretion of cytokines (or interleukines) bythe above-mentioned cytotoxic T lymphocytes, particularly IL-2, IL-4 orγ interferon.

As the case may be, the above-mentioned epitopes are selected from thoseable to induce in vitro the appearance and the growth of cytotoxic Tlymphocytes from animal or human cells, particularly from peripheralblood mononucleated cells (PBMC), in the presence of factors necessaryfor the growth and differentiation of the cytotoxic T cells.

The polyepitopic protein fragments of the invention are moreovercharacterized in that they are adapted to contain CD4 epitopesrecognized by auxiliary T cells in association with the MCH molecules ofclass II, this property favoring the induction and maintenance of theCD8⁺ T cells recognizing the epitopes comprised in said fragments.

The present invention is illustrated with the help of FIGS. 1 and 2,showing respectively peptide sequences of the E6 and E7 proteins of thestrain 16 of the human papillomavirus (HPV 16), as well as thepolyepitopic fragments of the invention, and the epitopes within thesefragments.

The invention more particularly has for its object the polyepitopicfragments of the E6 and E7 protein of HPV, and more particularly thoseof the E6 protein shown in FIG. 1, or by SEQ ID NO: 2, or those of theE7 protein, shown in FIG. 2, or by SEQ ID NO: 11, of HPV 16,characterized in that they comprise a peptide sequence of about 15 toabout 30 amino acids, this peptide sequence containing the amino acidsequences of at least 3 different epitopes, and preferably at least 4different epitopes binding stably to HLA molecules of identical ordifferent type, when these epitopes are obtained by enzymaticdegradation of said peptide sequence, particularly in the proteasome,such that at least 4 HLA molecules of different types, and preferably atleast 5 HLA molecules of different types, bind to these epitopes, these4 or 5 HLA molecules being selected from those of type A1, A2, A3, A11,A24, A29, B7, B8, B18, B27, B35, B44, B51, and B62.

Preferably, the polyepitopic fragments according to the invention aresuch that the number of amino acids of their peptide sequence is greaterthan or equal to 17, and less than or equal to 30.

The invention relates more particularly to the polyepitopic fragments ofthe E6 protein of HPV defined above, characterized in that they comprisea peptide sequence of about 15 to 30 amino acids, this peptide sequencecontaining amino acid sequences of at least 5 different epitopes, andpreferably at least 6 different epitopes binding stably to HLA moleculesof identical or different type, when these epitopes are obtained byenzymatic degradation of said peptide sequence, particularly in theproteasome, such that at least 6 HLA molecules of different types, andpreferably at least 7 HLA molecules of different types, bind to theseepitopes, these 6 or 7 HLA molecules being selected from those of typeA1, A2, A3, A11, A24, A29, B7, B8, B18, B27, B35, B44, and B51.

Preferably, the polyepitopic fragments of the E6 protein according tothe invention are such that the number of amino acids of their peptidesequence is greater than or equal to 20 (preferably greater than orequal to 22), and less than or equal to 30.

Also preferably, the above-mentioned polyepitopic fragments of the E6protein of HPV, are characterized in that they all comprise an epitopebinding to the HLA molecule of type B35, an epitope binding to the HLAmolecule of type B44, and an epitope binding to the HLA molecule of typeB51.

The invention more particularly has for its object the polyepitopicfragment of the E6 protein of HPV as defined above, characterized inthat it corresponds to the fragment of 30 amino acids delimited by theamino acids located in positions 15 and 44 of the peptide sequence ofthe E6 protein of HPV, and characterized by the peptide sequence SEQ IDNO: 4 as follows:

(15)RPRKLPQLCTELQTTIHDIILECVYCKQQL(44)

said fragment containing 9 epitopes binding stably to at least one ofthe 8 HLA molecules of the following types: A2, A11, A29, B7, B8, B35,B44, or B51, said epitopes being the following:

(15)RPRKLPQL(22) binding stably to HLA molecules of the B7 or B35 type,

(18)KLPQLCTEL(26) binding stably to HLA molecules of the A2 type,

(19)LPQLCTEL(26) binding stably to HLA molecules of the B51 type,

(21)QLCTELQTTI(30) binding stably to HLA molecules of the A2 type,

(24)TELQTTIHDI(33) binding stably to HLA molecules of the A29 or B44type,

(29)TIHDIILRCV(38) binding stably to HLA molecules of the A2 type,

(33)IILECVYCK(41) binding stably to HLA molecules of the A11 type,

(35)LECVYCKQQL(44) binding stably to HLA molecules of the A29 or B44type,

(37)CVYCKQQL(44) binding stably to HLA molecules of the B8 type.

The invention also relates to the polyepitopic fragment of the E6protein of HPV as defined above, characterized in that it corresponds tothe fragment of 17 amino acids delimited by the amino acids located atpositions 46 and 62, or to the fragment of 22 amino acids delimited bythe amino acids located at positions 46 and 67 of the peptide sequenceof the E6 protein of HPV, this latter fragment being characterized bythe peptide sequence SEQ ID NO: 6 as follows:

(46)RREVYDFAFRDLCIVYRDGNPY(67)

said fragment containing 6 epitopes binding stably to at least one ofthe 10 HLA molecules of the following types: A2, A3, A11, A24, A29, B7,B27, B35, B44, or B51, said epitopes being the following:

(46)RREVYDFAFR(55) binding stably to HLA molecules of the B27 type,

(49)VYDFAFRDL(57) binding stably to HLA molecules of the A24 type,

(50)YDFAFRDL(57) binding stably to HLA molecules of the A29 or B44 type,

(52)FAFRDLCIV(60) binding stably to HLA molecules of the A2, B35, B51,or B7 type,

(54)FRDLCIVYR(62) binding stably to HLA molecules of the A3 or A11 type,

(59)VYRDGNPY(67) binding stably to HLA molecules of the A3 or A11 type.

The invention also has for its object the polyepitopic fragment of theE6 protein of HPV as defined above, characterized in that it correspondsto the fragment of 29 amino acids delimited by the amino acids locatedat positions 80 and 108 of the peptide sequence of the E6 protein ofHPV, this latter fragment being characterized by the peptide sequenceSEQ ID NO: 8 as follows:

(80)ISEYRHYCYSLYGTTLEQQYNKPLCDLLI(108)

said fragment containing 6 epitopes binding stably to at least 10 HLAmolecules of the following types: A1, A3, A11, A24, A29, B7, B18, B35,B44, or B51, said epitopes being the following:

(80)ISEYRHYCY(88) binding stably to HLA molecules of the A1 or B18 type,

(81)SEYRHYCY(88) binding stably to HLA molecules of the A29 or B44 type,

(87)CYSLYGTTL(95) binding stably to HLA molecules of the A24 type,

(94)TLEQQYNK(101) binding stably to HLA molecules of the A3 or A11 type,

(95)LEQQYNKPL(103) binding stably to HLA molecules of the A29 or B44type,

(110)KPLCDLLI(108) binding stably to HLA molecules of the B7, B35 or B51type.

The invention more particularly has for its object the polyepitopicfragment of the E6 protein of HPV as defined above, characterized inthat it corresponds to the fragment of 22 amino acids delimited by theamino acids located at positions 118 and 139 of the peptide sequence ofthe E6 protein of HPV, this latter fragment being characterized by thepeptide sequence SEQ ID NO: 10 as follows:

(118)CPEEKQRHLDKKQRFHNIRGRW(139)

said fragment containing 6 epitopes binding stably to at least one ofthe 7 HLA molecules of the following types: A24, B8, B18, B27, B35, B44,or B51, said epitopes being the following:

(118)CPEEKQRHL(126) binding stably to HLA molecules of the B8, B18, B35,BS51 type,

(119)PEEKQRHL(126) binding stably to HLA molecules of the B44 type,

(127)DKKQRFHNI(135) binding stably to HLA molecules of the B8 type,

(128)KKQRFHNIR(136) binding stably to HLA molecules of the B27 type,

(130)QRFHNIRGRW(139) binding stably to HLA molecules of the B27 type,

(131)RFHNIRGRW(139)binding stably to HLA molecules of the A24 type.

The invention also relates to the polyepitopic fragments of the E7protein of HPV as defined above, characterized in that they comprise apeptide sequence of about 15 to 30 amino acids, this peptide sequencecontaining the amino acid sequences of at least 3 different epitopes,and preferably of at least 4 different epitopes binding stably to HLAmolecules of the identical or different type, when these epitopes areobtained by enzymatic degradation of said peptide sequence, particularlyin the proteasome, such that at least 4 HLA molecules of differenttypes, and preferably at least 5 HLA molecules of different types bindto these epitopes, these 4 or 5 HLA molecules being selected from thoseof type A1, A2, A3, A11, A29, B7, B18, B35, B44, and B62.

Preferably, the polyepitopic fragments of the E7 protein according tothe invention are such that the number of amino acids of the peptidesequence is greater than or equal to 17, and less than or equal to 23.

Again preferably, the polyepitopic fragments of the E7 protein of theabove-mentioned HPV, are characterized in that they all comprise anepitope binding to the HLA molecule of type B44.

The invention more particularly has for its object the polyepitopicfragment of the E7 protein of HPV as defined above, characterized inthat it corresponds to the fragment of 23 amino acids delimited by theamino acids located in positions 3 and 25 of the peptide sequence of theE7 protein of HPV, this latter fragment being characterized by thepeptide sequence SEQ ID NO: 14 as follows:

(3)GDTPTLHEYMLDLQPETTDLYCY(25)

said fragment containing 5 epitopes binding stably to at least one ofthe 6 HLA molecules of the following type: A1, A2, B18, B35, B44 or B62,said epitopes being the following:

(3)GDTPTLHEY(11) binding stably to HLA molecules of the B44 type,

(5)TPTLHEYML(13) binding stably to HLA molecules of the B35 type,

(11)YMLDLQPETT(20) binding stably to HLA molecules of the A2 type,

(15)LQPETTDLY(23) binding stably to HLA molecules of the B62 type,

(16)QPETTDLYCY(25) binding stably to HLA molecules of the A1 or B18type.

The invention also relates to the polyepitopic fragment of the E7protein of HPV as defined above, characterized in that it corresponds tothe fragment of 17 amino acids delimited by the amino acids located inpositions 44 and 60 of the peptide sequence of the E7 protein of HPV,this latter fragment being characterized by the peptide sequence SEQ IDNO: 16 as follows:

(44)QAEPDRAHYNIVTFCCK(60)

said fragment containing 4 epitopes binding stably to at least one ofthe 6 HLA molecules of the following types: A1, A3, A11, A29, B7, B18,B35, or B44, said epitopes being the following:

(44)QAEPDRAHY(52) binding stably to HLA molecules of the A1 or B18 type,

(45)AEPDRAHY(52) binding stably to HLA molecules of the A29 or B44 type,

(46)EPDRAHYNIV(55) binding stably to HLA molecules of the B7 or B35type,

(53)NIVTFCCK(60) binding stably to HLA molecules of the A3 or A11 type.

The invention also has for its object the polyepitopic fragment of theE7 protein of HPV as defined above, characterized in that it correspondsto the fragment of 19 amino acids delimited by the amino acids locatedin positions 79 and 97 of the peptide sequence of the E7 protein of HPV,this latter fragment being characterized by the peptide sequence SEQ IDNO: 18 as follows:

(79)LEDLLMGTLGIVCPICSQK(97)

said fragment containing 4 epitopes binding stably to at least one ofthe 5 HLA molecules of the following types: A2, A3, A11, A29 or B44,said epitopes being the following:

(79)LEDLLMGTL(87) binding stably to HLA molecules of the A29 or B44type,

(82)LLMGTLGIV(90) binding stably to HLA molecules of the A2 type,

(86)TLGIVCPI(93) binding stably to HLA molecules of the A2 type,

(89)IVCPICSQK(97) binding stably to HLA molecules of the A3 or A11 type.

The invention also has for its object the polyepitopic fragments of thep53 human protein characterized in that they comprise a peptide sequenceof about 20 to about 35 amino acids, this latter containing amino acidsequences of at least three different epitopes binding stably to HLAmolecules of identical or different type, when these epitopes areobtained by enzymatic degradation of said peptide sequence, particularlyin the proteasome, such that at least 3 HLA molecules of different typeswill be recognized by said epitopes and will bind to these latter, these3 HLA molecules being selected from those of type A1, A2, A3, A24, B7,B8, B27, B35, B44 and B62.

The invention also relates to the polyepitopic fragments of the p53human protein mentioned above, characterized in that they comprise apeptide sequence of about 20 to about 35 amino acids, this lattercontaining the amino acid sequences of at least 5 different epitopes,and preferably of at least 6 different epitopes binding to HLA moleculesof identical or different type, such that at least 3 HLA molecules ofdifferent types, and preferably at least 4 HLA molecules of differenttypes will be recognized by said epitopes and will bind to these latter,these 3 or 4 HLA molecules being selected from those of type A2, A24,B27, B35, B44 and B62.

The invention more particularly has for its object the polyepitopicfragment of the p53 human protein as defined above, characterized inthat it corresponds to the fragment of 32 amino acids delimited by theamino acids located in positions 106 and 137 of the peptide sequence ofthe p53 protein, or to the fragment of 36 amino acids delimited by theamino acids in positions 102 and 137 of said peptide sequence, thislatter fragment being characterized by the following peptide sequence:

(102)TYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQL(137)

said fragment containing 6 epitopes binding stably to at least one ofthe 4 HLA molecules of the following types: A2, A24, B35 or B62, saidepitopes being the following:

(102)TYQGSYGFRL(111) binding stably to HLA molecules of the A24 type,

(105)GSYGFRLGFL(114) binding stably to HLA molecules of the B35 type,

(106)SYGFRLGFL(114) binding stably to HLA molecules of the A24 type,

(118)TAKSVTCTY(134) binding stably to HLA molecules of the B62 type,

(125)TYSPALNKMF(134) binding stably to HLA molecules of the A24 type,

(129)ALNKMFCQL(137) binding stably to HLA molecules of the B35 type.

The invention also has for its object the polyepitopic fragment of thep53 human protein as defined above, characterized in that it correspondsto the fragment of 21 amino acids delimited by the amino acids locatedin the positions 149 and 169 of the peptide sequence of the p53 protein,and characterized by the following peptide sequence:

(149)STPPPGTRVRAMAIYKQSQHM(169)

said fragment containing 6 epitopes binding stably to at least one ofthe 6 HLA molecules of the following types: A2, A3, A24, B27, B35 orB62, said epitopes being the following:

(149)STPPPGTRV(157) binding stably to HLA molecules of the A2 type,

(152)PPGTRVRAM(160) binding stably to HLA molecules of the B35 type,

(155)TRVRAMAIYK(164) binding stably to HLA molecules of the B27 type,

(156)RVRAMAIY(163) binding stably to HLA molecules of the B62 type,

(156)RVRAMAIYK(164) binding stably to HLA molecules of the A3 type,

(162)IYKQSQHM(169) binding stably to HLA molecules of the A24 type.

The invention also relates to the polyepitopic fragment of the p53 humanprotein as defined above, characterized in that it corresponds to thefragment of 26 amino acids delimited by the amino acids located inpositions 187 and 212 of the peptide sequence of the p53 protein, or tothe fragment of 34 amino acids delimited by the amino acids located inpositions 187 and 220 of said peptide sequence, this latter fragmentbeing characterized by the following peptide sequence:

(187)GLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPY(220)

said fragment containing 11 epitopes binding stably to at least one ofthe 7 HLA molecules of the following types: A1, A2, A24, B7, B8, B27 orB44, said epitopes being the following:

(187)GLAPPQHLIRV(197) binding stably to HLA molecules of the A2 type,

(189)APPQHLIRV(197) binding stably to HLA molecules of the B7 type,

(195)IRVEGNLRVEY(205) binding stably to HLA molecules of the B27 type,

(196)RVEGNLRVEY(205) binding stably to HLA molecules of the A2 type,

(197)VEGNLRVEY(205) binding stably to HLA molecules of the B44 type,

(201)LRVEYLDDR(209) binding stably to HLA molecules of the B27 type,

(203)VEYLDDRNTF(212) binding stably to HLA molecules of the B44 type,

(204)EYLDDRNTF(212) binding stably to HLA molecules of the A24 type,

(210)NTFRHSVVV(218) binding stably to HLA molecules of the B8 type,

(211)TFRHSVVV(218) binding stably to HLA molecules of the A24 type,

(212)FRHSVVVPY(220) binding stably to HLA molecules of the B27 type.

The invention also has for its object the polyepitopic fragment of thep53 human protein as defined above, characterized in that it correspondsto the fragment of 18 amino acids delimited by the amino acids locatedin positions 226 and 243 of the peptide sequence of the p53 protein, andcharacterized by the following peptide sequence:

(226)GSDCTTIHYNYMCNSSCM(243)

said fragment containing 3 epitopes binding stably to at least one ofthe 3 HLA molecules of the following types: A1, A24 or B44, saidepitopes being the following:

(226)GSDCTTIHY(234) binding stably to HLA molecules of the A1 type,

(227)SDCTTIHYNY(236) binding stably to HLA molecules of the B44 type,

(235)NYMCNSSCM(243) binding stably to HLA molecules of the A24 type.

The invention also relates to the polyepitopic fragment of the p53 humanprotein as defined above, characterized in that it corresponds to thefragment of 25 amino acids delimited by the amino acids located inpositions 249 and 273 of the peptide sequence of the p53 protein, or tothe fragment of 26 amino acids delimited by the amino acids located inpositions 248 and 273 of said peptide sequence, or to the fragment of 33amino acids delimited by the amino acids located in positions 248 and280 of said peptide sequence, this latter fragment being characterizedby the following peptide sequence:

 (248)RRPILTIITLEDSSGNLLGRNSFEVRVCACPGR(280)

said fragment containing 8 epitopes binding stably to at least one ofthe 6 HLA molecules of the following types: A2, B7, B27, B35, B44 orB62, said epitopes being the following:

(248)RRPILTIITL(257) binding stably to HLA molecules of the B27 type,

(249)RPILTIITL(257) binding stably to HLA molecules of the B35 and B7type,

(255)ITLEDSSGN(263) binding stably to HLA molecules of the A2 type,

(257)LEDSSGNLL(265) binding stably to HLA molecules of the B44 type,

(263)NLLGRNSF(270) binding stably to HLA molecules of the B62 type,

(264)LLGRNSFEV(272) binding stably to HLA molecules of the A2 type,

(266)GRNSFEVR(273) binding stably to HLA molecules of the B27 type,

(272)VRVCACPGR(280) binding stably to HLA molecules of the B27 type.

The invention also relates to peptide sequences derived from thepolyepitopic fragments mentioned above, of the E6 or E7 proteins, or ofthe p53 protein, particularly:

by substitution and/or suppression and/or addition of one or severalamino acids, of the above-mentioned fragments, and/or

by modification of at least one peptide linkage —CO—NH— of the peptidechain of the above-mentioned fragments, particularly by introduction ofa retro or retro-inverso type linkage, and/or

by substitution of at least one amino acid of the peptide chain of thesequence or of the above-mentioned fragment, with a non-proteinogenicamino acid,

said derived sequences containing peptides or pseudopeptides bindingspecifically to the same molecule or molecules of MCH as those bindingto the peptides contained in the polyepitopic fragments mentioned abovefrom which they derive.

By derived sequence by introduction of a retro-inverso linkage, shouldbe understood any peptide analog of an above-mentioned fragment, saidanalog being constituted by a peptide chain in which at least one of theresidues on the one hand is bound to at least one adjacent residue by an—NH—CO— linkage, and on the other hand, is of a chirality opposite thatof the same amino acyl residue in the peptide chain of the parentpeptide (namely of the above-mentioned fragment from which it derives).

By a sequence derived by introduction of a retro linkage, should beunderstood any peptide analog of an above-mentioned fragment, saidanalog being constituted by a peptide chain in which at least one of theresidues is bound to at least one adjacent residue by an —NH—CO—linkage, the chirality of the whole of the amino acyl residues involvedin at least one —NH—CO— linkage being conserved relative to thecorresponding residues of the peptide chain of the parent peptide.

It follows that the —CO—NH— and —NH—CO— linkages must be taken intoaccount in the preceding, in the direction of the parent peptide chaingoing from the amino terminal (N-terminal) end toward the carboxyterminal (C-terminal) end.

By “proteinogenic amino acid”, is meant, in the preceding, any aminoacid entering into the constitution of a natural protein or peptide.

By “non-proteinogenic amino acid” is meant, in contrast to the precedingdefinition, any amino acid that does not enter into the constitution ofa natural protein or peptide. There is meant more particularly by“non-proteinogenic amino acid”, any amino acid whose carbon carrying a Rside chain, namely the —CHR— group, located between —CO— and —NH— in thenatural peptide chain, is replaced by a structure that does not enterinto the constitution of a natural protein or peptide.

The invention more particularly has for its object the derived sequencesas described above, characterized in that at least one of the peptidelinkages —CO—NH— of the peptide chain of the parent peptide is replacedby a linkage different from the —CO—NH— linkage, said different linkagebeing particularly selected from the following.

—CH₂—NH— (amino methylene); —CH₂—CH₂— (carba); —CO—CH₂— (cetomethylene);—CH₂—O— (methylene-oxy); —CHOH—CH₂— (hydroxyethylene); —CHOH—CHOH— (di-hydroxyethylene); —CH═CH— (E or Z olefin); —CHCN—NH— (aminocyanomethylene); —S—CH₂— (thiomethylene); —CH₂—S— (thio methylene);—CS—NH— (thioamide); —PO₂—NH— (phosphonamide); —CHOH—(hydroxymethylene); —NH—CO—NH— (urea);

(oxiran);

(tetrazole); —CH₂—CO—NH— (β-homologation); —CHOH—CH₂—NH— (aminohydroxyethylene); —CO—NH—NH— (hydrazino).

The invention also has for its object nucleotide sequences coding for apolyepitopic fragment of the E6 or E7 protein, or for a derived peptidesequence, as defined above, said nucleotide sequences being derived fromthe sequence SEQ ID NO: 1 coding for the E6 protein, or from thesequence SEQ ID NO: 11 coding for the E7 protein.

In this connection, the invention more particularly has for its objectthe nucleotide sequences defined above, selected from the following:

the sequence SEQ ID NO: 3, coding for the polyepitopic fragment SEQ IDNO: 4 mentioned above, of the E6 protein,

the sequence SEQ ID NO: 5, coding for the polyepitopic fragment SEQ IDNO: 6 mentioned above, of the E6 protein,

the sequence SEQ ID NO: 7, coding for the polyepitopic fragment SEQ IDNO: 8 mentioned above, of the E6 protein,

the sequence SEQ ID NO: 9, coding for the polyepitopic fragment SEQ IDNO: 10 mentioned above, of the E6 protein,

the sequence SEQ ID NO: 13, coding for the polyepitopic fragment SEQ IDNO: 14 mentioned above, of the E7 protein,

the sequence SEQ ID NO: 15, coding for the polyepitopic fragment SEQ IDNO: 16 mentioned above, of the E7 protein,

the sequence SEQ ID NO: 17, coding for the polyepitopic fragment SEQ IDNO: 18 mentioned above, of the E7 protein.

The invention also has for its object the nucleotide sequences codingfor a polyepitopic fragment of the p53 protein, or for a derived peptidesequence, as defined above.

The invention also has for its object any vector, particularly aplasmid, cosmid or phage, containing at least one above-mentionednucleotide sequence under the control of elements necessary for thetranscription of said sequence, particularly under the control of atranscription promoter and terminator.

The invention also relates to host cells, particularly bacterial, virus,yeasts, eucaryotic cells, transformed with the help of a vectormentioned above according to the invention, so as to integrate stablyinto their genome or to maintain in a stable manner in their cytoplasm,at least one nucleotide sequence according to the invention.

The invention also relates to any vector comprising one or severalpolyepitopic fragments and/or one or several derived peptide sequencesas defined above, or any vector comprising one or severalabove-mentioned nucleotide sequences, said vectors being selected fromthose adapted to ensure protection of said fragments or nucleotidesequences in the organism and/or their penetration into the cells of theorganism.

In the case of the use of polyepitopic fragments and/or theabove-mentioned derived peptide sequences, such vectors are selectedfrom fatty acids (in the framework of the preparation of lipopeptides),liposomes, etc.

In this connection, the invention more particularly has for its objectany lipopeptide characterized in that it comprises:

a peptide portion comprising one or several polyepitopic proteinfragments selected from those defined above, or any peptide sequencederived from said fragments as defined above,

and one or several lipophilic portions, preferably selected from thosecomprising:

a C4 to C20 hydrocarbon chain, saturated or unsaturated, linear orbranched,

or a steroid group, as the case may be connected to the above-mentionedhydrocarbon chain,

said lipophilic portions being if desired associated with a shortpeptide vector (thereby to form lipopeptide vector structures)comprising one or several ionized functions at physiological pH, and afunction permitting the covalent bonding of said hydrocarbon chainand/or said steroid group.

By lipophilic portion, in what precedes and what follows, is intendedany lipophilic molecule, insoluble in water, permitting, when it islinked to the peptide portion defined above, an intracellular passivepassage of the obtained lipopeptide, thanks to the hydrophobicproperties of said molecule. Preferably the lipopeptide resulting fromthe linking of the lipophile portion to the peptide portion, is solublein water.

Preferably, the hydrocarbon chain of the lipophilic portions, isselected from the following:

palmitic acid,

oleic acid,

linoleic acid,

linolenic acid.

Also preferably, the steroid group of the lipophilic portion or portionsis selected from cholesterol derivatives such as cholest-5-enyl-3-oxyacetic acid, or cholest-5-enyl-3-oxycarbonic acid.

The invention more particularly has for its object any lipopeptide asdescribed above, characterized in that the lipophilic portion orportions are bonded covalently to one or several amino acids of thepeptide portion.

Preferably, the lypophilic portion or portions are bonded covalently tothe αNH₂ or εNH₂ function of a lysine located in the N terminal or Cterminal position of the peptide portion, or to the thiol function of acystein, or to any amino, alcohol or thiol function if desired added tothis peptide with a single spacer.

In this connection, the invention more particularly has for its objectany lipopeptide as defined above, in which the lipophilic portion orportions are represented by a group

N⁶⁰ -acetyl-Lysine N^(ε)(palmitoyl) (also designated by the abbreviationAc-K(Pam)).

The present invention also has for its object micelles ormicroaggregates of one or several different lipopeptides defined above.

Preferably, said micelles or microaggregates have a size less than about1 μm.

Preferably, the micelles or microaggregates according to the inventionare as obtained by dispersion of said lipopeptides in a concentratedacetic acid solution of about 80%, or any other solvent capable ofensuring molecular dispersion of the lipopeptides in solution.

In the case of the use of nucleotide sequences defined above accordingto the invention, the above-mentioned vectors are selected from theviruses, particularly the retroviruses, the adenoviruses and theassociated viruses (AAV Adeno Associated Virus).

The invention also has for its object antibodies directed against thepolyepitopic protein fragments or the epitopes or their derived peptidesequences (or analogs) as defined above, said antibodies being thoseobtained by immunization of an animal with at least one of theabove-mentioned complexes, said antibodies being adapted to form acomplex with these polyepitopic fragments or these epitopes or theiranalogs.

The antibodies according to the invention are polyclonal or monoclonalantibodies.

The polyclonal antibodies mentioned above are obtained by immunizationof an animal with at least one polyepitopic protein fragment or anepitope or an analog according to the invention, followed by therecovery of the desired antibodies in purified form, by removal of theserum of said animal, and separation of said antibodies from the otherconstituents of the serum, particularly by affinity chromatography on acolumn on which is fixed a specific antigen recognized by the antibody,particularly a polyepitopic protein fragment or an epitope or an analogaccording to the invention.

The monoclonal antibodies according to the invention can be obtained bythe hybridome technique whose general principle is set forth below.

In a first instance, an animal is immunized, generally a mouse (orculture cells in an in vitro immunization framework) with a polyepitopicprotein fragment or an epitope or an analog according to the invention,against which the B lymphocytes of the animal are then capable ofproducing antibodies. These antibody-producing lymphocytes are thenfused with “immortal” myelomatous cells (particularly of mice) to giverise to hybridomes. From the heterogeneous mixture of cells thusobtained, there is then carried out a selection of the cells capable ofproducing a particular antibody and of multiplying indefinitely. Thishybridome is multiplied in the form of clones, each leading to theproduction of a monoclonal antibody whose recognition propertiesrelative to the polyepitopic protein fragment or epitope or the like ofthe invention, can be tested for example with ELISA, by immunotransferin one or two dimensions, by immunofluorescence, or with the help of abiodetector. The monoclonal antibodies thus selected are then purifiedparticularly according to the affinity chromatography techniquedescribed above.

The invention also relates to the use of one or several above-mentionedantibodies for practicing a diagnostic method in vitro of theabove-mentioned pathologies.

In this connection, the invention also has for its object sets or kitscomprising said antibodies, for practicing a diagnostic method asdefined above.

The invention also relates to pharmaceutical compositions, or vaccines,characterized in that they comprise:

a)

at least one polyepitopic fragment of the E6 or E7 protein as definedabove,

and/or at least one peptide sequence derived from this fragment, asdefined above,

and/or at least one suitable vector, particularly lipopeptides and/ormicelles defined above, containing at least one polyepitopic fragmentmentioned above of the E6 or E7 protein, and/or at least one sequencementioned above derived from these fragments,

in association with a physiologically acceptable vehicle,

said polyepitopic protein fragment and/or its derived sequence being, asthe case may be, associated with one or several other exogenous epitopesrecognized by auxiliary T cells (also called CD4 or T helper epitopes),said epitopes being selected particularly from the following:

the peptide fragment delimited by amino acids located in positions 830and 846 of the peptide sequence of the tetanus toxin, said fragmentresponding to the following formula: QYIKANSKFIGITELKK,

hemagglutinin (Prevost-Blondel et al., 1995, J. Virol, 62, No. 12, pp8046-8055),

PADRE epitope (Alexander et al., 1994, Immunity, 1,751).

or b)

at least one nucleotide sequence as defined above, coding for anabove-mentioned polyepitopic fragment of the E6 or E7 protein,

and/or at least one nucleotide sequence coding for a peptide sequencederived from this fragment, as defined above,

the above-mentioned nucleotide sequences being adapted to be used alone,as minigenes,

and/or at least one above-mentioned suitable vector, selectedparticularly from the viruses such as defined above, containing at leastone above-mentioned nucleotide sequence,

in association with a physiologically acceptable vehicle,

or c)

antibodies defined above, directed against a polyepitopic fragment ofthe E6 or E7 protein, and/or against a peptide sequence derived fromthese fragments, as defined above, in association with a physiologicallyacceptable vehicle.

Preferably, the pharmaceutical compositions or vaccines mentioned aboveare present in a form administrable subcutaneously, particularly inseveral injections (preferably 3 injections) of about 500 μg of thepolyepitopic fragment in the lipopeptide form, at about one monthintervals.

The invention has more particularly for its object the use ofpolyepitopic fragments of the E6 or E7 protein defined above, or of theabove-mentioned derived peptide sequences, or the above-definednucleotide sequences, or the above-mentioned antibodies, or thelipopeptides defined above, for the preparation of a medication orvaccine for the prevention or treatment of pathologies connected withthe infection of individuals by human papillomavirus, such as cervicalintraepithelial neoplasias (CIN), the invasive cancer of the neck of theuterus, vulvar intraepithelial neoplasias (VIN).

The invention also relates to pharmaceutical compositions or vaccinescharacterized in that they comprise:

a)

at least one polyepitopic fragment of the p53 protein as defined above,

and/or at least one peptide sequence derived from this fragment, asdefined above,

and/or at least one suitable vector, particularly the lipopeptidesand/or micelles defined above, containing at least one above-mentionedpolyepitopic fragment of the p53 protein, and/or at least oneabove-mentioned sequence derived from these fragments,

in association with a physiologically acceptable vehicle,

said polyepitopic protein fragment and/or its derived sequence being, asthe case may be, associated with one or several other exogenous epitopesrecognized by the auxiliary T cells (also called CD4 or T helperepitopes), said epitopes being selected particularly from those definedabove,

or b)

at least one nucleotide sequence as defined above, coding for anabove-mentioned polyepitopic fragment of the p53 protein,

and/or at least one nucleotide sequence coding for a peptide sequencederived from this fragment, as defined above,

the above-mentioned nucleotide sequences being adapted to be used alone,as minigenes,

and/or at least one above-mentioned suitable vector, selectedparticularly from the viruses as defined above, containing at least oneabove-mentioned nucleotide sequence,

in association with a physiologically acceptable vehicle,

or c)

antibodies defined above, directed against a polyepitopic fragment ofthe p53 protein, and/or against a peptide sequence derived from thesefragments, as defined above, in association with a physiologicallyacceptable vehicle.

The invention also has for its object the use:

of at least one polyepitopic fragment of the p53 protein, selected fromthose defined above,

and/or at least one peptide sequence derived from this fragment, asdefined above,

or at least one nucleotide sequence, as defined above, coding for apolyepitopic fragment of the p53 protein, and/or for a peptide sequencederived from this fragment, as defined above,

for the preparation of a medication or vaccine adapted for theprevention or treatment of cancers, particularly of the breast, of thecolon, of the lung or of the bladder.

The invention also relates to peptides or epitopes of the E6 protein ofHPV selected from the following:

(19)LPQLCTEL(26) binding stably to HLA molecules of the B51 type,

(21)QLCTELQTTI(30) binding stably to HLA molecules of the A2 type,

(24)TELQTTIHDI(33) binding stably to HLA molecules of the A29 or B44type,

(33)IILECVYCK(41) binding stably to HLA molecules of the A11 type,

(35)LECVYCKQQL(44) binding stably to HLA molecules of the A29 or B44type,

(37)CVYCKQQL(44) binding stably to HLA molecules of the B8 type,

(46)RREVYDFAFR(55) binding stably to HLA molecules of the B27 type,

(49)VYDFAFRDL(57) binding stably to HLA molecules of the A24 type,

(50)YDFAFRDL(57) binding stably to HLA molecules of the A29, B44 type,

(52)FAFRDLCIV(60) binding stably to HLA molecules of the A2, B35, B51type,

(54)FRDLCIVYR(62) binding stably to HLA molecules of the A3, A11 type,

(59)IVYRDGNPY(67) binding stably to HLA molecules of the A3, A11 type,

(81)SEYRHYCY(88) binding stably to HLA molecules of the A29, B44 type,

(87)CYSLYGTTL(95) binding stably to HLA molecules of the A24 type,

(94)TLEQQYNK(101) binding stably to HLA molecules of the A3, A11 type,

(95)LEQQYNKPL(103) binding stably to HLA molecules of the A29, B44 type,

(101)KPLCDLLI(108) binding stably to HLA molecules of the B7, B35, B51type,

(118)CPEEKQRHL(126) binding stably to HLA molecules of the B8, B18, B35,B51 type,

(119)PEEKQRHL(126) binding stably to HLA molecules of the B44 type,

(127)DKKQRFHNI(135) binding stably to HLA molecules of the B8 type,

(128)KKQRFHNIR(136) binding stably to HLA molecules of the B27 type,

(130)QRFHNIRGRW(139) binding stably to HLA molecules of the B27 type,

(131)RFHNIRGRW(139) binding stably to HLA molecules of the A24 type.

The invention also relates to peptides or epitopes of the E7 protein ofHPV selected from the following:

(3)GDTPTLHEY(11) binding stably to HLA molecules of the B44 type,

(5)TPTLHEYML(13) binding stably to HLA molecules of the B35 type,

(15)LQPETTDLY(23) binding stably to HLA molecules of the B62 type,

(16)QPETMDLYCY(25) binding stably to HLA molecules of the A1, B18 type,

(45)AEPDRAHY(52) binding stably to HLA molecules of the A29, B44 type,

(46)EPDRAHYNIV(55) binding stably to HLA molecules of the B7 or B35type,

(53)NIVTFCCK(60) binding stably to HLA molecules of the A3, A11 type,

(79)LEDLLMGTL(87) binding stably to HLA molecules of the A29, B44 type,

(89)IVCPICSQK(97) binding stably to HLA molecules of the A3, A11 type.

The invention also relates to peptide sequences derived from theabove-mentioned peptides, said derived sequences, or the like, being asdefined above in the framework of sequences derived from thepolyepitopic protein fragments mentioned above.

The invention also has for its object the nucleotide sequences codingfor peptides of the E6 or E7 proteins mentioned above, namely,

the sequence delimited by the nucleotides located in positions 43 and 66of the sequence SEQ ID NO: 1, coding for (15)RPRKLPQL(22),

the sequence delimited by the nucleotides located in positions 52 and 78of the sequence SEQ ID NO: 1, coding for (18)KLPQLCTEL(26),

the sequence delimited by the nucleotides located in positions 55 and 78of the sequence SEQ ID NO: 1, coding for (19)LPQLCTEL(26),

the sequence delimited by the nucleotides located in positions 61 and 90of the sequence SEQ ID NO: 1, coding for (21 )QLCTELQTTI(30),

the sequence delimited by the nucleotides located in positions 70 and 99of the sequence SEQ ID NO: 1, coding for (24)TELQTTIHD(33),

the sequence delimited by the nucleotides located in positions 97 and123 of the sequence SEQ ID NO: 1, coding for (33)IILECVYCK(41),

the sequence delimited by the nucleotides located in positions 103 and132 of the sequence SEQ ID NO: 1, coding for (35)LECVYCKQQL(44),

the sequence delimited by the nucleotides located in positions 109 and132 of the sequence SEQ ID NO: 1, coding for (37)CVYCKQQL(44),

the sequence delimited by the nucleotides located in positions 136 and165 of the sequence SEQ ID NO: 1, coding for (46)RREVYDFAFR(55),

the sequence delimited by the nucleotides located in positions 145 and171 of the sequence SEQ ID NO: 1, coding for (49)VFDFAFRDL(57),

the sequence delimited by the nucleotides located in positions 148 and171 of the sequence SEQ ID NO: 1, coding for (50)YDFAFRDL(57),

the sequence delimited by the nucleotides located in positions 154 and180 of the sequence SEQ ID NO: 1, coding for (52)FAFRDLCIV(60),

the sequence delimited by the nucleotides located in positions 160 and186 of the sequence SEQ ID NO: 1, coding for (54)FRDLCIVYR(62),

the sequence delimited by the nucleotides located in positions 175 and201 of the sequence SEQ ID NO: 1, coding for (59)IVYRDGNPY(67),

the sequence delimited by the nucleotides located in positions 241 and264 of the sequence SEQ ID NO: 1, coding for (81)SEYRHYCY(88),

the sequence delimited by the nucleotides located in positions 259 and285 of the sequence SEQ ID NO: 1, coding for (87)CYRLYGTTL(95),

the sequence delimited by the nucleotides located in positions 280 and303 of the sequence SEQ ID NO: 1, coding for (94)TLEQQYNK(101),

the sequence delimited by the nucleotides located in positions 283 and309 of the sequence SEQ ID NO: 1, coding for (95)LEQQYNKPL(103),

the sequence delimited by the nucleotides located in positions 301 and324 of the sequence SEQ ID NO: 1, coding for (101)KPLCDLLI(108),

the sequence delimited by the nucleotides located in positions 352 and378 of the sequence SEQ ID NO: 1, coding for (1 18)CPEEKQRHL(126),

the sequence delimited by the nucleotides located in positions 355 and378 of the sequence SEQ ID NO: 1, coding for (119)PEEKQRHL(126),

the sequence delimited by the nucleotides located in positions 379 and405 of the sequence SEQ ID NO: 1, coding for (127)DKKQRFHNI(135),

the sequence delimited by the nucleotides located in positions 382 and408 of the sequence SEQ ID NO: 1, coding for (128)KKQRFHNIR(136),

the sequence delimited by the nucleotides located in positions 388 and417 of the sequence SEQ ID NO: 1, coding for (130)QRFHNIRGRW(139),

the sequence delimited by the nucleotides located in positions 391 and417 of the sequence SEQ ID NO: 1, coding for (131)RFHNIRGRW(139),

the sequence delimited by the nucleotides located in positions 7 and 33of the sequence SEQ ID NO: 2, coding for (3)GDTPTLHEY(11),

the sequence delimited by the nucleotides located in positions 13 and 39of the sequence SEQ ID NO: 2, coding for (5)TPTLHEYML(13),

the sequence delimited by the nucleotides located in positions 43 and 69of the sequence SEQ ID NO: 2, coding for (15)LQPETTDLY(23),

the sequence delimited by the nucleotides located in positions 46 and 75of the sequence SEQ ID NO: 2, coding for (16)QPETTDLYCY(25),

the sequence delimited by the nucleotides located in positions 133 and153 of the sequence SEQ ID NO: 2, coding for (45)AEPDRAHY(52),

the sequence delimited by the nucleotides located in positions 136 and165 of the sequence SEQ ID NO: 2, coding for (46)EPDRAHYNIV(55),

the sequence delimited by the nucleotides located in positions 157 and180 of the sequence SEQ ID NO: 2, coding for (53)NIVTFCCK(60),

the sequence delimited by the nucleotides located in positions 235 and261 of the sequence SEQ ID NO: 2, coding for (79)LEDLLMGTL(87),

the sequence delimited by the nucleotides located in positions 265 and291 of the sequence SEQ ID NO: 2, coding for (89)IVCPICSQK(97).

The invention also has for its object epitopes of the p53 proteinselected from the following:

(102)TYQGSYGFRL(111) binding stably to HLA molecules of the A24 type,

(105)GSYGFRLGFL(114) binding stably to HLA molecules of the B35 type,

(106)SYGFRLGFL(114) binding stably to HLA molecules of the A24 type,

(118)TAKSVTCTY(126) binding stably to HLA molecules of the B62 type,

(125)TYSPALNKMF(134) binding stably to HLA molecules of the A24 type,

(152)PPGTRVRAM(160) binding stably to HLA molecules of the B35 type,

(155)TRVRAMAIYK(164) binding stably to HLA molecules of the B27 type,

(156)RVRAMAIY(163) binding stably to HLA molecules of the B62 type,

(162)IYKQSQHM(169) binding stably to HLA molecules of the A24 type,

(195)IRVEGNLRVEY(205) binding stably to HLA molecules of the B27 type,

(197)VEGNLRVEY(205) binding stably to HLA molecules of the B44 type,

(201)LRVEYLDDR(209) binding stably to HLA molecules of the B27 type,

(203)VEYLDDRNTF(212) binding stably to HLA molecules of the B44 type,

(204)EYLDDRNTF(212) binding stably to HLA molecules of the A24 type,

(211)TFRHSVVV(218) binding stably to HLA molecules of the A24 type,

(212)FRHSVVVPY(220) binding stably to HLA molecules of the B27 type,

(227)SDCTTIHYNY(236) binding stably to HLA molecules of the B44 type,

(235)NYMCNSSCM(243) binding stably to HLA molecules of the A24 type,

(249)RPILTIITL(257) binding stably to HLA molecules of the B35 type,

(257)LEDSSGNLL(265) binding stably to HLA molecules of the B44 type,

(263)NLLGRNSF(270) binding stably to HLA molecules of the B62 type,

(266)GRNSFEVR(273) binding stably to HLA molecules of the B27 type,

(272)VRVCACPGR(280) binding stably to HLA molecules of the B27 type.

The invention also has for its object any process for the preparation ofpolyepitopic fragments, of single epitopes (above-mentioned peptides),or of derived sequences, by conventional peptide synthesis in liquid orsolid phase.

As a modification, the polyepitopic fragments, single epitopes orderived peptide sequences, as defined above according to the invention,can be obtained in the form of recombinant polypeptides bytransformation of suitable host cells as defined above with the help ofvectors containing a recombinant nucleotide sequence as defined aboveaccording to the invention, and the recovery, as the case may be afterpurification, of the recombinant polypeptide coded by said nucleotidesequence and produced by the host cells mentioned above.

24 1 477 DNA Human Papillomavirus CDS (1)..(477) 1 atg cac caa aag agaact gca atg ttt cag gac cca cag gag cga ccc 48 Met His Gln Lys Arg ThrAla Met Phe Gln Asp Pro Gln Glu Arg Pro 1 5 10 15 aga aag tta cca cagtta tgc aca gag ctg caa aca act ata cat gat 96 Arg Lys Leu Pro Gln LeuCys Thr Glu Leu Gln Thr Thr Ile His Asp 20 25 30 ata ata tta gaa tgt gtgtac tgc aag caa cag tta ctg cga cgt gag 144 Ile Ile Leu Glu Cys Val TyrCys Lys Gln Gln Leu Leu Arg Arg Glu 35 40 45 gta tat gac ttt gct ttt cgggat tta tgc ata gta tat aga gat ggg 192 Val Tyr Asp Phe Ala Phe Arg AspLeu Cys Ile Val Tyr Arg Asp Gly 50 55 60 aat cca tat gct gta tgt gat aaatgt tta aag ttt tat tct aaa att 240 Asn Pro Tyr Ala Val Cys Asp Lys CysLeu Lys Phe Tyr Ser Lys Ile 65 70 75 80 agt gag tat aga cat tat tgt tatagt ttg tat gga aca aca tta gaa 288 Ser Glu Tyr Arg His Tyr Cys Tyr SerLeu Tyr Gly Thr Thr Leu Glu 85 90 95 cag caa tac aac aaa ccg ttg tgt gatttg tta att agg tgt att aac 336 Gln Gln Tyr Asn Lys Pro Leu Cys Asp LeuLeu Ile Arg Cys Ile Asn 100 105 110 tgt caa aag cca ctg tgt cct gaa gaaaag caa aga cat ctg gac aaa 384 Cys Gln Lys Pro Leu Cys Pro Glu Glu LysGln Arg His Leu Asp Lys 115 120 125 aag caa aga ttc cat aat ata agg ggtcgg tgg acc ggt cga tgt atg 432 Lys Gln Arg Phe His Asn Ile Arg Gly ArgTrp Thr Gly Arg Cys Met 130 135 140 tct tgt tgc aga tca tca aga aca cgtaga gaa acc cag ctg tga 477 Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg GluThr Gln Leu 145 150 155 2 158 PRT Human Papillomavirus 2 Met His Gln LysArg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro 1 5 10 15 Arg Lys LeuPro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp 20 25 30 Ile Ile LeuGlu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu 35 40 45 Val Tyr AspPhe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly 50 55 60 Asn Pro TyrAla Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile 65 70 75 80 Ser GluTyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu 85 90 95 Gln GlnTyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn 100 105 110 CysGln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys 115 120 125Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met 130 135140 Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 145 150 1553 90 DNA Human Papillomavirus CDS (1)..(90) 3 cga ccc aga aag tta ccacag tta tgc aca gag ctg caa aca act ata 48 Arg Pro Arg Lys Leu Pro GlnLeu Cys Thr Glu Leu Gln Thr Thr Ile 1 5 10 15 cat gat ata ata tta gaatgt gtg tac tgc aag caa cag tta 90 His Asp Ile Ile Leu Glu Cys Val TyrCys Lys Gln Gln Leu 20 25 30 4 30 PRT Human Papillomavirus 4 Arg Pro ArgLys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile 1 5 10 15 His AspIle Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu 20 25 30 5 66 DNA HumanPapillomavirus CDS (1)..(66) 5 cga cgt gag gta tat gac ttt gct ttt cgggat tta tgc ata gta tat 48 Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg AspLeu Cys Ile Val Tyr 1 5 10 15 aga gat ggg aat cca tat 66 Arg Asp Gly AsnPro Tyr 20 6 22 PRT Human Papillomavirus 6 Arg Arg Glu Val Tyr Asp PheAla Phe Arg Asp Leu Cys Ile Val Tyr 1 5 10 15 Arg Asp Gly Asn Pro Tyr 207 87 DNA Human Papillomavirus CDS (1)..(87) 7 att agt gag tat aga cattat tgt tat agt ttg tat gga aca aca tta 48 Ile Ser Glu Tyr Arg His TyrCys Tyr Ser Leu Tyr Gly Thr Thr Leu 1 5 10 15 gaa cag caa tac aac aaaccg ttg tgt gat ttg tta att 87 Glu Gln Gln Tyr Asn Lys Pro Leu Cys AspLeu Leu Ile 20 25 8 29 PRT Human Papillomavirus 8 Ile Ser Glu Tyr ArgHis Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu 1 5 10 15 Glu Gln Gln TyrAsn Lys Pro Leu Cys Asp Leu Leu Ile 20 25 9 66 DNA Human PapillomavirusCDS (1)..(66) 9 tgt cct gaa gaa aag caa aga cat ctg gac aaa aag caa agattc cat 48 Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg PheHis 1 5 10 15 aat ata agg ggt cgg tgg 66 Asn Ile Arg Gly Arg Trp 20 1022 PRT Human Papillomavirus 10 Cys Pro Glu Glu Lys Gln Arg His Leu AspLys Lys Gln Arg Phe His 1 5 10 15 Asn Ile Arg Gly Arg Trp 20 11 297 DNAHuman Papillomavirus CDS (1)..(297) 11 atg cat gga gat aca cct aca ttgcat gaa tat atg tta gat ttg caa 48 Met His Gly Asp Thr Pro Thr Leu HisGlu Tyr Met Leu Asp Leu Gln 1 5 10 15 cca gag aca act gat ctc tac tgttat gag caa tta aat gac agc tca 96 Pro Glu Thr Thr Asp Leu Tyr Cys TyrGlu Gln Leu Asn Asp Ser Ser 20 25 30 gag gag gag gat gaa ata gat ggt ccagct gga caa gca gaa ccg gac 144 Glu Glu Glu Asp Glu Ile Asp Gly Pro AlaGly Gln Ala Glu Pro Asp 35 40 45 aga gcc cat tac aat att gta acc ttt tgttgc aag tgt gac tct acg 192 Arg Ala His Tyr Asn Ile Val Thr Phe Cys CysLys Cys Asp Ser Thr 50 55 60 ctt cgg ttg tgc gta caa agc aca cac gta gacatt cgt act ttg gaa 240 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp IleArg Thr Leu Glu 65 70 75 80 gac ctg tta atg ggc aca cta gga att gtg tgcccc atc tgt tct cag 288 Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys ProIle Cys Ser Gln 85 90 95 aaa cca taa 297 Lys Pro 12 98 PRT HumanPapillomavirus 12 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met LeuAsp Leu Gln 1 5 10 15 Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln LeuAsn Asp Ser Ser 20 25 30 Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly GlnAla Glu Pro Asp 35 40 45 Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys LysCys Asp Ser Thr 50 55 60 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp IleArg Thr Leu Glu 65 70 75 80 Asp Leu Leu Met Gly Thr Leu Gly Ile Val CysPro Ile Cys Ser Gln 85 90 95 Lys Pro 13 69 DNA Human Papillomavirus CDS(1)..(69) 13 gga gat aca cct aca ttg cat gaa tat atg tta gat ttg caa ccagag 48 Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu 15 10 15 aca act gat ctc tac tgt tat 69 Thr Thr Asp Leu Tyr Cys Tyr 20 1423 PRT Human Papillomavirus 14 Gly Asp Thr Pro Thr Leu His Glu Tyr MetLeu Asp Leu Gln Pro Glu 1 5 10 15 Thr Thr Asp Leu Tyr Cys Tyr 20 15 51DNA Human Papillomavirus CDS (1)..(51) 15 caa gca gaa ccg gac aga gcccat tac aat att gta acc ttt tgt tgc 48 Gln Ala Glu Pro Asp Arg Ala HisTyr Asn Ile Val Thr Phe Cys Cys 1 5 10 15 aag 51 Lys 16 17 PRT HumanPapillomavirus 16 Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val ThrPhe Cys Cys 1 5 10 15 Lys 17 57 DNA Human Papillomavirus CDS (1)..(57)17 ttg gaa gac ctg tta atg ggc aca cta gga att gtg tgc ccc atc tgt 48Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 1015 tct cag aaa 57 Ser Gln Lys 18 19 PRT Human Papillomavirus 18 Leu GluAsp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 10 15 SerGln Lys 19 36 PRT Homo sapiens 19 Thr Tyr Gln Gly Ser Tyr Gly Phe ArgLeu Gly Phe Leu His Ser Gly 1 5 10 15 Thr Ala Lys Ser Val Thr Cys ThrTyr Ser Pro Ala Leu Asn Lys Met 20 25 30 Phe Cys Gln Leu 35 20 21 PRTHomo sapiens 20 Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala IleTyr Lys 1 5 10 15 Gln Ser Gln His Met 20 21 34 PRT Homo sapiens 21 GlyLeu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg 1 5 10 15Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val 20 25 30Pro Tyr 22 18 PRT Homo sapiens 22 Gly Ser Asp Cys Thr Thr Ile His TyrAsn Tyr Met Cys Asn Ser Ser 1 5 10 15 Cys Met 23 33 PRT Homo sapiens 23Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn 1 5 1015 Leu Leu Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly 20 2530 Arg 24 17 PRT Artificial Sequence Description of Artificial SequenceSynthetic tetanus toxin peptide sequence 24 Gln Tyr Ile Lys Ala Asn SerLys Phe Ile Gly Ile Thr Glu Leu Lys 1 5 10 15 Lys

What is claimed is:
 1. A polyepitope of E6 protein from HPV, comprising 17 amino acids delimited by amino acids located in positions 46 and 62, or to 22 amino acids delimited by amino acids located in positions 46 and 67 of the peptide sequence of the E6 protein of HPV, characterized by the peptide sequence SEQ ID NO: 6 as follows: (46)RREVYDFAFRDLCIVYRDGNPY(67) said peptide sequence contains 6 epitopes binding stably to at least one of the 10 HLA molecules of the following types: A2, A3, A11, A24, A29, B7, B27, B35, B44, or B51, said epitopes being the following: (46)RREVYDFAFR(55) binding stably to HLA molecules of the B27 type, (49)VYDFAFRDL(57) binding stably to HLA molecules of the A24 type, (50)YDFAFRDL(57) binding stably to HLA molecules of the A29 or B44 type, (52)FAFRDLCIV(60) binding stably to HLA molecules of the A2, B35, B51, or B7 type, (54)FRDLCIVYR(62) binding stably to HLA molecules of the A3 or All type, and (59)IVYRDGNPY(67) binding stably to HLA molecules of the A3 or All type.
 2. A polyepitope comprising amino acid 46 and 62 of SEQ ID NO:
 6. 3. A polyepitope comprising amino acids 46 and 67 of SEQ ID NO:
 6. 4. A polyepitope consisting essentially of SEQ ID NO:
 6. 